Process for enzymatic replacement of the B-30 amino acid in insulins

ABSTRACT

The B-30 amino acid in insulins is replaced enzymatically by 
     reacting as substrate component the selected insulin Ins-X, wherein X represents the B-30 amino acid 
     with an amine component selected from the group consisting of 
     (a) amino acids of the formula 
     
         H--B--OH 
    
     wherein B is an amino acid residue, 
     (b) optionally N-substituted amino acid amides of the formula 
     
         H--B--NR.sup.1 R.sup.2 
    
     wherein B is an amino acid residue and R 1  and R 2  are independently selected from the group consisting of hydrogen, amino, hydroxy, alkyl, cycloalkyl, aryl, heteroaryl and aralkyl or R 1  and R 2  together with the nitrogen atom form a heterocyclic group which may contain a further hetero atom, and 
     (c) amino acid esters of the formula 
     
         H--B--OR.sup.3, H--B--SR.sup.3 or H--B--SeR.sup.3 
    
     wherein B is am amino acid residue and R 3  represents alkyl, cycloalkyl, aryl, heteroaryl or aralkyl 
     in the presence of an L-specific serine or thiol carboxypeptidase enzyme, preferably carboxypeptidase-Y, in an aqueous solution or dispersion having a pH from about 7 to 10.5, thereby to form an insulin derivative 
     Ins--B--OH, Ins--B--NR 1  R 2 , Ins--B--B--NR 1  R 2 , 
     Ins--B--OR 3 , Ins--B--SR 3  or Ins--B--SeR 3   
     and subsequently cleaving a group --NR 1  R 2 , --B--NR 1  R 2 , --OR 3 , --SR 3  or SeR 3 , if desired, preferably by using a carboxypeptidase enzyme. The cleaving may also be performed on derivatives obtained by other methods. 
     By using porcine insulin as substrate component and threonine as the amino acid forming part of the amine component human insulin is obtained.

This application is a continuation-in-part of U.S. Ser. No. 136,661,filed Apr. 2, 1980 by Jack T. Johansen and Fred Widmer; U.S. Ser. No.136,661 issued as U.S. Pat. No. 4,339,534 on July 13, 1982, and wasco-pending with this application.

BACKGROUND OF THE INVENTION

1. Field of the invention

The present invention generally relates to a process for enzymaticreplacement of the C-terminal amino acid in the B-chain (B-30) ofinsulins from various species.

It is well known that insulins from different vertebrate speciesincluding mammals and humans differ in their primary structure. SinceSanger in 1958 determined the primary structure of bovine insulin theprimary structure of insulins from other vertebrate species has beendetermined.

These results as summarized in the below figure using the porcineinsulin as model indicate that amino acid substitutions can occur atmany positions within either chain. However, certain structural featuresare common to all the insulins, e.g. the position of the 3 disulfidebonds, the N-terminal region of the A-chain, the B23-26 sequence in theC-terminal region of the B-chain, etc.

The differences in the primary structure of some common insulins areseen from the below table:

    ______________________________________                                               A-chain         B-chain                                                       4    8       9      10    3    29    30                                ______________________________________                                        Bovine   Glu    Ala     Ser  Val   Asn  Lys   Ala                             Sheep    Glu    Ala     Gly  Val   Asn  Lys   Ala                             Horse    Glu    Thr     Gly  Ileu  Asn  Lys   Ala                             Sei whale                                                                              Glu    Ala     Ser  Thr   Asn  Lys   Ala                             Porcine  Glu    Thr     Ser  Ileu  Asn  Lys   Ala                             Sperm whale                                                                            Glu    Thr     Ser  Ileu  Asn  Lys   Ala                             Dog      Glu    Thr     Ser  Ileu  Asn  Lys   Ala                             Human    Glu    Thr     Ser  Ileu  Asn  Lys   Thr                             Rabbit   Glu    Thr     Ser  Ileu  Asn  Lys   Ser                             Rat 1    Asp    Thr     Ser  Ileu  Lys  Lys   Ser                             Rat 2    Asp    Thr     Ser  Ileu  Lys  Met   Ser                             ______________________________________                                    

While the invention is described more fully below with relation to thespecific conversion of porcine insulin into human insulin, viz.replacement of B-30 alanine by threonine, it will easily be understoodthat the described method applies equally well to other types of insulinin that e.g. rabbit insulin may also be converted into human insulin,bovine insulin may be converted into B-30 (Thr) bovine insulin, etc.

2. Background of invention, especially with relation to conversion ofporcine insulin into human insulin

The idea of converting porcine insulin into human insulin bysemi-synthetic procedure has been an attractive problem in the field ofinsulin chemistry.

As stated above, human insulin differs from porcine insulin by only oneamino acid, the C-terminal residue of the B-chain (B-30) being threoninein human and alanine in porcine insulin, respectively. The exchange ofalanine to threonine was initially performed chemically and recentlyenzymatic procedures have been used. Ruttenberg (1972) (Ref. 1) hasdescribed the chemical conversion of porcine insulin into human insulin:esterification to insulin-hexamethylester, hydrolysis with trypsin todesoctapeptide insulin (DOI)-pentamethylester, blocking of the aminoterminal residues, chemical coupling with a synthetic octapeptide of thecorresponding human insulin sequence, acidic deprotection of theamino-groups, and finally alkaline saponifcation of the methyl estergroups. However, nobody has ever been able to produce pure human insulinby this method, since the chemical procedures, and in particular thefinal alkaline saponification steps seriously damage the insulinmolecule and also isoasparagine at the C-terminal residue of the A-chainis formed (Gattner et al. (Ref. 2)). To prevent this effect Obermeierand Geiger (1976) (Ref. 3) have carried out the fragment condensationwithout protection of the side chain carboxyl groups of DOI. They couldisolate human insulin after extensive purification, but only in very lowyields. Similar approaches have been taken by Gattner et al. (Ref. 2),using various insulin fragments. However, using the chemical methodsnobody has so far prepared pure human insulin in more than traceamounts.

M. Bodanszky et al. provides a process for preparing human insulin inU.S. Pat. No. 3,276,961 wherein human insulin was ostensibly preparedfrom other animal insulins by an action of an enzyme such ascarboxypeptidase A and trypsin in the presence of threonine. Thisprocess is not likely to produce human insulin because trypsin andcarboxypeptidase A hydrolyze not only the peptide bond of lysyl-alanine(B29-B30) but also the other positions in insulin under the conditiondescribed there. Trypsin preferentially hydrolyzes the peptide bond ofarginyl-glycine (B22-B23) than that of lysyl-alanine (B29-B30).Meanwhile, carboxypeptidase A cannot release solely the alanine atC-terminal of the B chain without liberating asparagine at C-terminal ofthe A chain. A special condition, i.e. reacting in an ammoniumhydrogencarbonate buffer solution, is necessary to prevent the releaseof the asparagine. The condition was discovered in 1978 (Schmitt,Hoppe-Seyler's Z. Physiol. Chem., 359, 799-802 (1978)). Furthermore,peptide synthesis may hardly occur because hydrolysis ratio is fasterthan synthesis ratio in the condition. Inouye et al. (Ref. 4) have shownthat human insulin can be obtained by coupling N-terminal protected DOIfrom porcine insulin with a synthetic octapeptide corresponding toresidues B-23-B30 of human insulin using trypsin as a catalyst.

However, this method is cumbersome in that it requires firstly atrypsin-catalyzed digestion of porcine insulin to form DOI, which isN-terminal protected by acylation with BOC-N₃ and then incubated for 20h with a separately synthesized human B23-B30 octapeptide, wherein B29lysine is BOC-protected. The obtained (BOC)₃ -human insulin issubsequently deprotected with trifluoroacetic acid/anisole at 0° C. for60 min. The yield was 49% based on the (BOC)₂ --DOI used.

Similarly, Morihara et al. (Ref. 5) have synthesized human insulin fromdes-alanine (B-30)-insulin (DAI) obtained by digestion of porcineinsulin with carboxypeptidase A for 8 h. The DAI (10 mM) was incubatedwith a large excess (0,5M) of threonine--OBu^(t) ester at 37° C. for 20h in the presence of high concentrations of organic co-solvents. Theformed [Thr--OBu^(t) --B--30] insulin was then deprotected withtrifluoroacetic acid in the presence of anisole. The yield was 41%. In asimilar experiment [Thr--B--30] bovine insulin was obtained in 60%yield.

Also this method is cumbersome in that it requires a pretreatment of theinitial insulin, long coupling times and a separate deprotection step.Also high amounts of organic co-solvents are necessary to minimize thehydrolytic activity of the enzyme.

In a similar experiment Morihara et al. (Ref. 6) used AchromobacterProtease I as enzymatic catalyst for the coupling of DAI with a largeexcess of Thr--OBu^(t) under formation of [Thr--OBu^(t) --B--30]insulin, which was isolated and deprotected as above. Although highyields (52%) may be obtained the reaction time was 20 h.

A similar experiment with bovine insulin leads to [Thr--OBu^(t) --B--30]bovine insulin in 58% yield.

The processes disclosed in Ref. 5 and 6 are also described in EuropeanPatent Application No. EP 17938 published on Oct. 29, 1980 and DanishApplication No. 1556/80.

Recently, it has been demonstrated that the enzyme carboxypeptidase-Y isan effective catalyst in peptide synthesis (Widmer and Johansen (Ref. 7)and Danish Patent Application No. 1443/79, filed Apr. 6, 1979).Furthermore, it has been shown that the enzyme under certain conditionscatalyzes the exchange of the C-terminal amino acid in a peptide withanother amino acid or amino acid derivative in a transpeptidationreaction (cf. International Appln. No. PCT/DK80/00020, filed Apr. 1,1980 and published on Oct. 16, 1980 as WO 80/02151, and EuropeanApplication No. EP 17485, published on Oct. 15, 1980, U.S. applicationSer. No. 136,611 filed Apr. 2, 1980 and Ser. No. 220,022 filed Dec. 2,1980 and based on the above PCT/DK80/0020. The underlying reactionprinciples are more fully explained by the inventors Breddam et al.(Ref. 11) who also discovered the so far unrecognizedpeptidyl-amino-acid-amide hydrolase activity of CPD-Y. Theabove-mentioned applications and the inventor's articles areincorporated herein by reference.

Although the general principle of an enzyme catalyzed transpeptidationreaction is thus described and exemplified in the above PCT and U.S.applications, the applicability thereof in connection with insulins hasnot been mentioned or shown.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a process forenzymatic replacement of the B-30 amino acid in insulins, which is notentailed with the drawbacks mentioned in the foregoing, and moreparticularly a process that does not require pretreatment of the insulinstarting material, long reaction times and chemical deprotection steps.

More specifically it is an object of the invention to provide a uniqueand simple process for conversion of porcine insulin to human insulin inhigh purity and good yields.

Another object of the invention is to provide an improved process forcleaving B-30 carboxyl group protected insulin derivatives, particularlydeamidating amide derivatives of various insulins without regard totheir source.

Briefly this and other objects of the invention can be attained in aprocess for enzymatic replacement of the B-30 amino acid in insulinswhich comprises

reacting as substrate component the selected insulin Ins-X, wherein Xrepresents the B-30 amino acid

with an amine component selected from the group consisting of

(a) amino acids of the formula

    H--B--OH

wherein B is an amino acid residue,

(b) optionally N-substituted amino acid amides of the formula

    H--B--NR.sup.1 R.sup.2

wherein B is an amino acid residue and R¹ and R² are independentlyselected from the group consisting of hydrogen, amino, hydroxy, alkyl,cycloalkyl, aryl, heteroaryl and aralkyl or R¹ and R² together with thenitrogen atom form a heterocyclic group which may contain a furtherhetero atom, and

(c) amino acid esters of the formula

    H--B--OR.sup.3, H--B--SR.sup.3 or H--B--SeR.sup.3

wherein B is an amino acid residue and R³ represents alkyl, cycloalkyl,aryl, heteroaryl or aralkyl

in the presence of an L-specific serine or thiol carboxypeptidase enzymefrom yeast or of animal, vegetable or microbial origin in an aqueoussolution or dispersion having a pH from about 7 to 10.5, thereby to forman insulin derivative

Ins--B--OH, Ins--B--NR¹ R², Ins--B--B--NR¹ R²,

Ins--B--OR³, Ins--B--SR³ or Ins--B--Ser³

and subsequently cleaving a group --NR¹ R², B--NR¹ R², --OR³, --SR³ orSeR³, if desired.

The above-mentioned cleaving step is of course not limited to insulinderivatives prepared according to the first transpeptidation step, butmay be applied on any such derivative without regard to its source, inparticular derivatives prepared according to EP 17938 as shown below inexample 5.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is based on the surprising recognition that theabove-mentioned enzyme carboxypeptidase Y can convert porcine insulininto human insulin by exchanging B-30 alanine with threonine in a singlestep without isolation of an intermediate product and subsequentdeprotection treatment. Optionally an insulin amide intermediate may beisolated, and, if desired, subsequently deamidated, using the sameenzyme carboxypeptidase Y.

As further elucidated below, the most suitable threonine derivative forthis conversion is threonine amide. However, the conversion will oftenresult in a mixture of human insulin and a certain amount of unreactedporcine insulin which are difficult to separate, why the insulin amideintermediates formed are preferably separated from the reaction mixturei.a. containing unreacted porcine insulin, and subsequently deamidated,advantageously using the same enzyme carboxypeptidase Y, as also furtherexpounded below.

Due to the broad peptidase specificity of CPD-Y as thoroughly describedand shown in the patent applications and articles referred to above theinvention is not limited to porcine insulin as starting material, butany other insulin, e.g. from the species described earlier, and anyother amino acid may be used for the replacement reaction.

Also other enzymes are applicable as described below.

The simplicity of the process according to the invention is indeedsurprising, even on the background of the application PCT/DK80/00020,and U.S. Pat. No. 3,276,961 (Bodanszky), since it might well be expectedthat not only the B-30 amino acid, but also the A-21 Asparagine commonto all the above-mentioned known insulins would be attacked by theenzyme.

In the above-mentioned earlier application the desired enzymaticcharacteristics with regard to a general peptide synthesis are explainedin detail, and it is stated that a plurality of carboxypeptidasesexhibit different enzymatic activities which are very dependent on pH sothat e.g. in a basic environment at a pH from 8 to 10.5 they displaypredominantly esterase or amidase activity and at a pH from 9 to 10.5 noor only insignificant carboxypeptidase activity, which, however, becomesmore and more pronounced with pH-values decreasing below 9. The esteraseactivity is less important in the present context but otherwise theseproperties can be advantageously used in the process of the invention,because they contribute to the achievement of a one step process withgood yields.

The applicable carboxypeptidases in the process of the invention areL-specific serine or thiol carboxypeptidases. Such enzymes can beproduced by yeast fungi, or they may be of animal, vegetable ormicrobial origin.

A particularly expedient enzyme is carboxypeptidase Y from yeast fungi(CPD-Y). This enzyme is described in the earlier applications i.a. withreference to Johansen et al. (Ref. 8) who developed a particularlyexpedient purification method by affinity chromatography on an affinityresin comprising a polymeric resin matrix with coupled benzylsuccinylgroups. CPD-Y, which is a serine enzyme, is characterized by having theabove relation between the different enzymatic activities at pH 9 and byhaving no endopeptidase activity. Another advantage of CPD-Y is that itis available in large amounts and displays relatively great stability.Further details are given in Ref. 7 and 11.

In addition to CPD-Y, which is the preferred enzyme at present, theprocess of the invention is feasible with other carboxypeptidases, suchas those listed in the following survey:

    ______________________________________                                        Enzyme            Origin                                                      ______________________________________                                                          Fungi                                                       Penicillocarboxypeptidase S-1                                                                   Penicillium janthinellum                                    Penicillocarboxypeptidase S-2                                                                   Penicillium janthinellum                                    Carboxypeptidase(s) from                                                                        Aspergillus saitoi                                          Carboxypeptidase(s) from                                                                        Aspergillus oryzae                                                            Plants                                                      Carboxypeptidase(s) C                                                                           Orange leaves                                                                 Orange peels                                                Carboxypeptidase C.sub.N                                                                        Citrus natsudaidai Hayata                                   Phaseolain        French bean leaves                                          Carboxypeptidase(s) from                                                                        Germinating barley                                                            Germinating cotton plants                                                     Tomatoes                                                                      Watermelons                                                                   Bromelain(pineapple)powder                                  ______________________________________                                    

The close relationship between a number of the above carboxypeptidasesis discussed by Kubota et al. (Ref. 12).

The process of the invention can in principle be carried out with anynatural, semi-synthetic or synthetic insulin as substrate component.

It should be mentioned that ionizable groups which are present in theindividual amino acids, which are constituents of the insulin startingmaterial, if desired, may be blocked in a manner known per se, dependingupon the type of the group. However, this is absolutely not necessary,which is precisely one of the advantages of the present process. If forsome reason it should be desired to protect the functional groups,suitable protective groups may be found in the above-mentionedapplications, in particular WO 80/02151.

The second participant in the reaction is the so-called amine componentwhich is selected from the group consisting of

(a) amino acids of the formula

    H--B--OH

wherein B is an amino acid residue,

(b) optionally N-substituted amino acid amides of the formula

    H--B--NR.sup.1 R.sup.2

wherein B is an amino acid residue and R¹ and R² are independentlyselected from the group consisting of hydrogen, amino, hydroxy, alkyl,cycloalkyl, aryl, heteroaryl, and aralkyl or R¹ and R² together with thenitrogen atom form a heterocyclic group which may contain a furtherhetero atom, and

(c) amino acid esters, thioesters or selenoesters of the formula

    H--B--OR.sup.3, H--B--SR.sup.3 or H--B--SeR.sup.3

wherein B is an amino acid residue and R³ represents alkyl, cycloalkyl,aryl, heteroaryl or aralkyl.

The amino acid forming part of the amine component may be any of theknown amino acids, e.g. leu, ile, ala, gly, ser, val, thr, lys, arg,asn, glu, gln, met, phe, tyr, trp or his.

In this context "alkyl" means straight chain or branched alkyl,preferably with 1 to 6 carbon atoms, e.g. methyl, ethyl, propyl,isopropyl, butyl, isobutyl, tert.butyl, amyl, hexyl and the like.

"Cycloalkyl" preferably means C₃ -C₈ cycloalkyl, e.g. cyclopropyl,cyclobutyl, etc.

"Aryl" is preferably phenyl and the like.

"Aralkyl" means benzyl, phenethyl, and the like. As stated the groups R¹and R² may be the same or different.

"Heteroaryl" as well as the heterocyclic group which may be formed byR¹, R² and the nitrogen atom are represented by e.g. pyridyl,pyrrolidyl, pyrimidinyl, morpholinyl, pyrazinyl, imidazolyl, etc.

All of these groups may be substituted with substituents which are inertwith relation to the enzyme, e.g. halo (fluoro, chloro, bromo, iodo),nitro, alkoxy (methoxy, ethoxy, etc.), or alkyl (methyl, ethyl, etc.).

Thus in case of all types of esters the group OR³ is preferably selectedfrom alkoxy groups, such as methoxy, ethoxy or t-butoxy, phenyloxy, andbenzyloxy groups. The groups may optionally be substituted with inertsubstituents, such as nitro groups (p-nitrobenzyloxy).

It is seen that in case of amides, when R¹ =hydrogen, R² =hydrogenrepresents the free amide, while R² =OH is a hydroxamic acid, R² =aminois a hydrazide, and R² =phenyl represents an anilide.

As stated above, the process of the invention is carried out at pH 7.0to 10.5, preferably at pH 7.5 to 10.5. The preferred pH-value, which isoften within a very narrow range, depends upon the pH-optima andpH-minima, respectively, for the different enzymatic activities of theenzyme used, it being understood that the pH-value should be selected sothat the activities are counterbalanced as explained in the foregoing.

If CPD-Y is used as enzyme, the pH-value is preferably 7.5 to 10.5,particularly 9.0 to 10.5, as explained below. However, a low pH withinthe preferred range such as about 7.5 is particularly expedient, if anisolation of insulin amide intermediates is desired.

The selected pH-value should preferably be maintained throughout thecoupling reaction, and may then be changed for precipitation of thereaction product, cleavage of protective groups, etc. A pH might beselected at which the enzyme displays amidase activity therebypreventing precipitation of the formed insulin amide and thuscontributes advantageously to the formation of the desired insulin inone step. Also a pH might be selected where the enzyme displayspredominantly peptidase activity thereby favouring the formation ofstable insulin amide intermediates.

The pH-control may be provided for by incorporating a suitable bufferfor the selected pH-range in the reaction medium, such as a bicarbonatebuffer.

The pH-value may also be maintained by adding an acid, such as HCl, or abase, such as NaOH, during the reaction. This may conveniently be doneby using a pH-stat.

Based on the information given above and in Ref. 7 and 11, the skilledperson will be able to select the most suitable reaction conditions,especially with regard to the pH, by which the various enzymaticactivities (amidase, peptidase, esterase, carboxypeptidase andpeptidyl-amino-acid-amide hydrolase) might best be utilized dependingupon the insulin substrate component, the amine component and theintention to suppress or favour the formation of intermediates.

Generally speaking low pH-values within the above range favour theformation and precipitation of an insulin amide intermediate, whilehigher values lead to a cleaving of the amide group due to the morepronounced amidase activity of the carboxypeptidase enzyme.

However, these conditions may also be influenced upon by varying theenzyme concentration, reaction time, etc.

The reaction is, as mentioned, carried out in an aqueous reaction mediumwhich, if desired, may contain up to 50% by volume of an organicsolvent. Preferred organic solvents are alkanols, e.g. methanol andethanol, glycols, e.g. ethylene glycol or polyethylene glycols, dimethylformamide, dimethyl sulfoxide, tetrahydrofurane, dioxane anddimethoxyethane.

The selection of the composition of the reaction medium dependsparticularly upon the solubility, temperature and pH of the reactioncomponents and the insulin products involved and upon the stability ofthe enzyme.

The reaction medium may also comprise a component that renders theenzyme insoluble, but retains a considerable part of the enzymeactivity, such as an ion exchanger resin. Alternatively, the enzyme maybe immobilized in known manner, cf. Methods of Enzymology, Vol. 44,1976, e.g. by bonding to a matrix, such as a cross-linked dextran oragarose, or to a silica, polyamide or cellulose, or by encapsulating inpolyacrylamide, alginates or fibres. Besides, the enzyme may be modifiedby chemical means to improve its stability or enzymatic properties.

In case it is desired to suppress any precipitation of insulin amideintermediates, the reaction medium may also contain urea or guanidinehydrochloride in concentrations up to 3 molar. This may also beadvantageous at pH-values and in media where the insulin substratecomponent has a limited solubility.

The concentration of the two participants in the reaction may varywithin wide limits, as explained below. A preferred startingconcentration for the insulin substrate component is 0.002 to 0.05 molarand for the amine component 0.05 to 3 molar.

The enzyme activity may vary as well, but the concentration ispreferably 10⁻⁶ to 10⁻⁴ molar, in particular 10⁻⁵ molar. The mostadvantageous activity depends i.a. on the substrate concentration, theamine concentration and the reaction time.

According to the invention the reaction temperature is preferably 20° to40° C. The most appropriate reaction temperature for a given synthesiscan be determined by experiments, but depends particularly upon the usedamine component and enzyme concentration. An appropriate temperaturewill usually be about 20° to 30° C., preferably about 25° C. Attemperatures lower than 20° C. the reaction time will usually beinappropriately long, while temperatures above 40° C. often causeproblems with the stability of the enzyme and/or reactants or of thereaction products.

Similar variations occur for the reaction time which depends very muchupon the other reaction parameters, especially the enzyme concentration.The standard reaction time in the process of the invention is about 2-6hours.

It should be added that when using an amide or substituted amide as theamine component, it is normally desired to cleave the amide groupspecifically from the formed insulin amide. Also in this respect thecarboxypeptidase, especially CPD-Y is very suitable since as describedabove CPD-Y exhibits amidase activity at pH>9 while the carboxypeptidaseactivity is negligible.

By the same token the carboxypeptidase might generally be used to cleavethe ester groups OR³, SR³ or SeR³ as defined from the formed insulinester intermediate to obtain a final insulin which is not C-terminalprotected.

Before the process of the invention will be illustrated by examples,starting materials, methods of measurement, etc. will be explained ingeneral terms.

Starting Materials

Porcine insulin was kindly supplied by Nordisk Insulinlaboratorium,Cophenhagen. Carboxypeptidase Y from baker's yeast, a commercialpreparation of the Carlsberg Breweries, was isolated by a modificationof the affinity chromatographic procedure of Johansen et al. (Ref. 8)and obtained as a lyophilized powder (10% enzyme in sodium citrate).Before use the enzyme was desalted on a "Sephadex G-25" column (1.5×25cm) equilibrated and eluted with water. The concentration of the enzymewas determined spectrophotometrically using E₂₈₀ nm^(1%) =14.8. Theenzyme preparation used was free of Protease A as checked by the assayof Lee and Riordan (Ref. 9). L-threonine amide was purchased fromVega-Fox, Ariz., USA. L-threonine methyl ester from Fluka, Switzerland,and L-threonine, Dansyl chloride, carboxypeptidase A and trypsin wereobtained from Sigma, USA. Chromatographic materials were products ofPharmacia, Sweden. All other reagents and solvents were analytical gradefrom Merck, W. Germany.

Amino Acid Analyses

Samples for amino acid analysis were hydrolyzed in 6M HCl at 110° C. invacuum for 24 hours, and analyzed on a Durrum D-500 amino acid analyzer.The amino acid compositions were based on the known content of asparticacid and glycine. Thr, Lys and Ala are the only amino acids affected bythe reactions. The values of these amino acids for porcine (human)insulin are: Thr=1.93 (2.87), Lys=0.97 (0,98) and Ala=2.00 (1.05). Theamount of unconverted porcine insulin in the reaction mixture wasdetermined from the alanine analysis of the insulin pool afterchromatography on "Sephadex G-50". The coupling yield is defined as theamount of a given product divided with the amount of all insulinconsumed in the reaction.

Carboxypeptidase A Digestions (examples 2 and 3 below)

To 100 μl of a solution of insulin or insulin derivatives (0.7 mg/ml) in0.1M Tris-HCl, pH 7.5, was added 10 μg of carboxypeptidase A. Afterdigestion at room temperature for 6 hours the reaction was stopped byaddition of an equal volume of 0.5M HCl, and the release of amino acidsdetermined by amino acid analysis.

Enzymatic digestion of insulin derivatives (example 4)

Digestion of various insulin derivatives with carboxypeptidases A and Ywere performed in a 0.5M Tris buffer, pH 7.5 at room temperature usingapproximately 1.5 mM insulin and 5 μM carboxypeptidase. Reaction timeswere 3 hours with CPD-A and 1.5 hours with CPD-Y. Under these conditionsmaximal release of C-terminal amino acids were obtained. Afteracidification with HCl, the aliquots were applied directly on the aminoacid analyzer.

The sequence of the C-terminal portion of the various insulinderivatives were determined after trypsin digestion and reaction of thedigest with Dansyl chloride, followed by identification of the Dansylpeptides. Digestion of insulin derivatives with trypsin was performed in0.1M NaHCO₃ at pH 8.2, using 1 mM insulin, 40 μM DPCC-trypsin and anincubation time of 1 hour. Preliminary experiments on porcine insulinhad indicated that these conditions were sufficient for complete releaseof the C-terminal alanyl residue from the B-chain. The released aminoacids or dipeptides were dansylated as follows: A 100 μl sample of thetryptic digestion mixture was quenched by addition of 100 μl 0.5M HCl.The aliquots were evaporated to dryness and redissolved in 100 μl 0.1MNaHCO₃, pH 8.2, 100 μl Dansyl chloride (5 mg/ml) in acetone was added,and the reaction mixture incubated for 2 hours at 37° C. The reactionmixture was then analyzed by HPLC, using the Waters liquidchromatography system, consisting of a Model U6K injector, two Model6000 A pumps, a Model 660 Solvent Programmer, a Model 450 UV detector, aWaters Data Module and a Waters Radial Compression Module (RCM 100)housing equipped with a Waters Radial Pak A (C-18 reverse phase) column.

The following standard compounds were synthesized: Dns--Ala--OH,Dns--Thr--OH, Dns--Ala--Thr--OH, Dns--Thr--Thr--OH, Dns--Thr--NH₂,Dns--Thr--Thr--NH₂ and Dns--Ala--NH₂. Using the procedure describedabove for the dansylation of insulin digestions, three of thesederivatives could by synthesized from H--Ala--OH, H--Thr--OH andH--Thr--NH₂. The dansylated dipeptides were synthesized viaDns--Ala--OMe and Dns--Thr--OMe: 1 mmol H--Ala--OMe•HCl was dissolved in0.1M NaHCO₃, 5 mmol of Dansyl chloride was added, and the reactionmixture incubated for 2 hours. The Dns--Ala--OMe was extracted from thereaction mixture with ethyl acetate and evaporated to dryness. It wasdemonstrated by HPLC that the isolated material was pure. The sameprocedure was used to synthesize Dns--Thr--OMe. Using procedures forenzymatic peptide synthesis similar to those previously described (Ref.7 and 11) these two compounds were then coupled to H--Thr--OH, givingDns--Ala--Thr--OH and Dns--Thr--Thr--OH and H--Thr--NH₂, givingDns--Ala--Thr--NH₂ and Dns--Thr--Thr--NH₂. The conditions were asfollows: 5 mM substrate, 0.5M nucleophile, 0.1M KCl, 2 mM EDTA, 1 μmCPD-Y, pH 9.0, 10% ethanol. All seven Dansyl derivatives could readilybe separated by HPLC using two different programs.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings,

FIG. 1 shows the reaction course when porcine insulin is reacted withL-Threonine amide using CPD-Y as catalyst. The amino acid analysis isplotted vs. the reaction time.

FIG. 2 shows the similar reaction course when L-Threonine methyl esteris used as amine component.

FIG. 3 shows an elution profile from an ion exchange chromatography onthe product of reaction between porcine insulin and threonine amide.

EXAMPLE 1 (Background investigations)

Porcine insulin was incubated with carboxypeptidase-Y at 25° C. and pH5-7, a pH-range where the enzyme exhibits maximal peptidase activity.Thereby the following amino acids from the C-terminal of the B-chainwere released: 1.0 Alanine, 1.0 Lysine, 1.0 Proline, 1.0 Threonine, 1.0Tyrosine, and 2.0 Phenylalanine. At position B-23 (glycine)carboxypeptidase-Y stops, and hence complete release of the first sevenamino acids in the B-chain of insulin can be achieved. Surprisingly, theC-terminal asparagine (A-21) of the A-chain is not released at all. AtpH 9.5 the C-terminal alanine of the B-chain is released much fasterthan the following amino acids. Importantly, the Leu--Tyr (B15-16) bondwas not hydrolyzed since the purified CPD-Y preparations used were freeof protease A (endopeptidase) in contrast to many preparationscommercially available from other sources, cf. Ref. 11.

EXAMPLE 2 (conversion of porcine insulin into human insulin usingthreonine amide as amine component without isolation of insulin amideintermediates)

To a solution of zinc-free porcine insulin (2 mM) in 10 mM EDTA, 0.1MKCL containing 0.5M L-Threonine amide at pH 9.5 and 25° C. was addedcarboxypeptidase-Y (50 μM). The pH of the reaction was kept constant byaddition of 0.5M NaOH using a pH-stat. To follow the reaction aliquotsat various time were taken and the reaction stopped by addition of 6MHCl to bring the pH to 1-2. The sample was then chromatographed on"Sephadex®G-50" (1×30 cm) equilibrated with 1M acetic acid in order toseparate insulin from the enzyme and free amino acids. The insulincontaining fractions were then lyophilized, and the amino acidcomposition determined as described above.

The reaction course is shown in FIG. 1. From the amino acid analysis itwas calculated that the threonine content increased by 0.7 moles permole of insulin, whereas 0.8 moles of alanine was lost together with 0.2moles of lysine (FIG. 1). These results suggested that after 6.5 hoursof reaction 20% of the porcine insulin was unreacted, while 20% of theinsulin had in addition to the alanine also lost the next amino acidlysine. The loss of 0.8 moles of alanine was accompanied byincorporation of 0.7 moles of threonine.

The reaction products were further analyzed by carboxypeptidase Adigestions. Carboxypeptidase A liberates only amino acids with freeα-carboxyl groups and does not liberate lysine. In accordance with thisspecificity carboxypeptidase A digestion of porcine insulin onlyreleases alanine and asparagine from the C-terminal ends of the B-chainand A-chain, respectively. Hence incubation of the insulin sampleobtained after 6.5 hours of reaction (FIG. 1) should in addition toasparagine only release alanine in an amount corresponding to thefraction of unreacted porcine insulin. Surprisingly, besides alaninealso threonine was released in amounts equivalent to the threonine amideincorporated to form the intermediate human insulin amide. This must bedue to the fact that in addition to its peptidase and esterase activitycarboxypeptidase-Y also has peptide amide hydrolase activity, cf. Ref.11. Apparently, during the reaction of porcine insulin in the presenceof carboxypeptidase-Y the C-terminal alanine is initially exchanged withthreonine amide in a transpeptidation reaction to form human insulinamide, which then is hydrolyzed by carboxypeptidase-Y to give humaninsulin in a total yield of about 60%, 20% unreacted porcine insulin and20% are other break down products. Other experiments have confirmed thissequence of reactions. Using less enzyme or shorter reaction time,carboxypeptidase A digestion of the reaction products showed lessthreonine release compared to threonine incorporation i.e. predominanthuman insulin amide was formed.

To separate the reaction products, the sample obtained after 6.5 hoursof reaction (FIG. 1) was subjected to high performance liquidchromatography using a "Lichrosorb RP-18", 5 μm, reverse phase column(0.4×30 cm) and a Waters Model 6000A pump and Model 450 UV detectoroperating at 220 nm. The eluent was 28.75% CH₃ CN in 5 mM tartratebuffer pH 3.0 containing 5 mM nBu--SO₃ Na and 50 mM Na₂ SO₄ (cf. Inouye,Ref. 4). The flow rate was 1.0 ml/min. Using this system human andporcine insulin could not be separated but all other by-products wereremoved. The amino acid analysis of the chromatographed material isshown in Table I. The analysis is in excellent agreement with thecomposition expected from the theoretical values. The content of 2.6moles of threonine and 1.3 moles of alanine suggests that the samplecontained about 70% human and 30% porcine insulin.

                  TABLE I                                                         ______________________________________                                        Amino Acid Composition of Insulin                                                    Moles Amino Acid                                                                             Sequence                                                Amino Acid                                                                             Mole Insulin     Porcine  Human                                      ______________________________________                                        Aspartic acid                                                                          3.1              3        3                                          Threonine                                                                              2.6              2        3                                          Serine   3.1              3        3                                          Glutamic acid                                                                          7.2              7        7                                          Proline  1.2              1        1                                          Glycine  4.0              4        4                                          Alanine  1.3              2        1                                          Valine   3.5*             4        4                                          Isoleucine                                                                             1.4*             2        2                                          Leucine  6.1              6        6                                          Tyrosine 3.6              4        4                                          Phenylalanine                                                                          3.0              3        3                                          Histidine                                                                              1.9              2        2                                          Lysine   1.0              1        1                                          Arginine 1.0              1        1                                          ______________________________________                                         *Low values due to incomplete hydrolysis                                 

EXAMPLE 3 (Conversion of porcine insulin into human insulin usingthreonine methylester as amine component without isolation ofintermediates)

To a solution of zinc-free porcine insulin (8 mM) in 10 mM EDTA, 0.1MKCl containing 0.5M-L-Threonine methylester at pH 9.5 and 25° C. wasadded carboxypeptidase-Y (60 μM). The pH of the reaction was keptconstant by addition of 0.5M NaOH using a pH-stat. To follow thereaction aliquots at various time were taken and the reaction stopped byaddition of 6M HCl to bring the pH to 1-2. The sample was thenchromatographed on "Sephadex G-50" (1×30 cm) equilibrated in 1M aceticacid in order to separate insulin from the enzyme and free amino acids.The insulin containing fractions were then lyophilized, and the aminoacid composition determined as described above.

The reaction course is shown in FIG. 2. The results of this reaction arevery similar to those described above using threonine amide: Threonineis incorporated concomitantly with the release of alanine and a smallamount of lysine. Carboxypeptidase A digestion of the sample after 17hours of reaction liberated both alanine and threonine, suggesting thatthe C-terminal alanine of the B-chain is first exchanged with threoninemethylester to form human insulin monomethylester, which is subsequentlyhydrolyzed by carboxypeptidase-Y to give human insulin. While thereaction qualitatively is similar to that described above for threonineamide, the final reaction mixture contains only 40% human insulin, 40%of the porcine insulin was not converted and 20% was hydrolyzed to otherproducts.

EXAMPLE 4 (Conversion of porcine insulin into human insulin usingthreonine amide as amine component and with isolation of intermediateinsulin amides).

To a solution of zinc free porcine insulin (2 mM) in 2 mM EDTA, 0.1M KCland 1.5M guanidine hydrochloride and containing 0.5M threonine amide(L--Thr--NH₂) at pH 7.5 anf 25° C. was added CPD-Y (15 μM). The pH ofthe reaction was kept constant by addition of 0.5M NaOH using a pH-stat.To follow the reaction course aliquots at various times were taken andthe reaction was quenched after 2 hours by adjusting the pH to 1.5-2.0with 1M HCl. The insulin fraction was separated from the enzyme and lowmolecular weight compounds by chromatography on "Sephadex®G-50 fine"(1×30 cm) equilibrated with 1M acetic acid and was lyophilized. Aminoacid analysis on the lyophilized "insulin pool" is described aboveindicated that 78% of the porcine insulin had been consumed in thereaction.

In order to further analyse the reactants present in the "insulin pool"ion exchange chromatography on "DEAE-Sephadex®A-25" was performedessentially as described by Morihara et al (Ref. 5). The lyophilizedinsulin sample (75 mg) was dissolved in 0.01M Tris, 2.5M urea, 0.05MNaCl, pH 7.5 and applied to a "DEAE Sephadex®A-25" column (0.5×25 cm)equilibrated with the same buffer. The insulin was eluted with a NaClgradient from 0.05 to 0.30M in the same buffer and 8 ml fractions werecollected on a "Sephadex®G-25" column and lyophilized.

The elution profile is shown in FIG. 3, where three peaks are observed.Also stated is the amino acid compositions of the individual peaks andthe peak compositions as a result of digestion experiments performedwith CPD-Y and CPD-A as described above. Since only the C-terminal ofthe B-chain of insulin is involved in these reactions,--Pro--Lys--Ala--OH is used as an abbreviation of porcine insulin. Otherinsulin derivatives are thus abbreviated accordingly:--Pro--Lys--Thr--OH=human insulin, --Pro--Lys--Thr--NH₂ =human insulinamide, etc. It is seen that at pH 7.5 which was used for the reaction,and where the amidase activity of CPD-Y generally is lower than thepeptidase activity, the peptide amide formed is sufficiently stablesince peak I comprised about 20% of the total insulin pool. In thereaction about 75% of the porcine insulin starting material has beenconverted. However, since the threonine content of peak I (3,65) islarger than the 3.0 in human insulin, it is evident that the initialtranspeptidation product (--Pro--Lys--Thr--NH₂) is not sufficientlystable to avoid an oligomerisation under formation of(--Pro--Lys--Thr--Thr--NH₂).

This formation of a mixture of amide intermediates might on the face ofit indicate that a homogenous human insulin would not be formed by theearlier described deamidation step by means of CPD-Y, since deamidationwould be expected to result in a mixture of --Pro--Lys--Thr--OH and--Pro--Lys--Thr--Thr--OH. However, when the peak I mixture was subjectedto a deamidation treatment with 10 μM CPD-Y at pH 10.0 in 0.1M HCl, 2 mMEDTA for 20 minutes, surprisingly almost pure human insulin wasobtained. The experiment proceeded as follows:

After separation of the reacted insulin from enzyme and low molecularweight material by chromatography on "Sephadex G-50", the material waschromatographed on "DEAE Sephadex A-25" using procedures identical tothose in FIG. 3. The reaction product eluted in Peak II as expected,while the unreacted material (<10%) eluted in Peak I. No Peak III waspresent, i.e. no insulin derivatives without lysine were formed. Asindicated from the results in Table II below obtained by CPD-A, CPD-Yand trypsin digestion, only the threonine content was significantlyaffected by these reactions. This is in agreement with the expectedabsence of peptidase activity of CPD-Y at this pH.

The amino acid composition of Peak II from the deamidation reaction isclose to the analysis of human insulin: Thr=2.87, Ala=1.05, Lys=0.98.The digestion of the deamidated product with CPD-Y, CPD-A and trypsinindicated that the sample contain 90-95% of pure human insulin. Thissuggests that the insulin derivative --Pro--Lys--Thr--Thr--NH₂ reactsalmost exclusively via the peptidyl-amino-acid-amide hydrolase activitywhile --Pro--Lys--Thr--NH₂ reacts mostly via the amidase activity. Theoverall yield for the conversion of porcine insulin to human insulin isapproximately 30% based on the amount of converted insulin.

                  TABLE II                                                        ______________________________________                                        Amino acid composition of Peaks I, II, and III from FIG. 3                    and Peak II obtained after deamidation treatment of                           Peak I fraction                                                                                                 Peak II ob-                                                                   tained after                                                                  deamidation                                         Peak I Peak II  Peak III  of Peak I                                   ______________________________________                                        Aspartic acid                                                                           2.99     2.99     3.01    3.01                                      Threonine 3.62     2.52     2.50    2.79                                      Serine    2.93     2.92     2.92    2.93                                      Glutamine acid                                                                          6.88     7.16     7.18    7.01                                      Proline   1.00     0.98     0.71    0.87                                      Glycine   4.00     4.00     4.00    4.00                                      Alanine   1.16     1.52     1.03    1.09                                      Valine    2.42     2.82     2.81    2.50                                      Ileucine  0.94     1.04     1.09    1.19                                      Leucine   5.80     6.21     6.18    6.10                                      Tyrosine  3.80     3.86     3.67    4.02                                      Phenylalanine                                                                           2.74     2.82     2.65    2.64                                      Histidine 1.96     1.90     1.93    1.89                                      Lysine    0.99     0.74     0.06    1.01                                      Arginine  0.96     1.00     0.98    0.89                                      ______________________________________                                    

EXAMPLE 5 (Deamidation of human insulin amide obtained bytrypsin-catalyzed condensation of Des-alanine (B30) porcine insulin(DAI) with threonine amide).

In order to substantiate that the advantageous CPD-Y catalyzeddeamidation of insulin amides is not limited to insulin amides obtainedby the transpeptidation process of the invention, human insulin amidewas prepared in accordance with the general teachings of Morihara et al.(EP 17 938 and Ref. 5 and 6). It is noted, however, that while amidesare mentioned in EP 17 938 as one among other protecting groups for thecarboxyl group in threonine, its applicability has not been proved byexamples, the only example dealing with threonine tert. butyl ester.

The reaction is illustrated by the following scheme: ##STR1##

This method provides the significant advantage over the methodexemplified in EP 17 938, that the enzymatic deamidation is much milderthan the acid-catalyzed deesterification according to Morihara et al.

Preparation of DAI--ThrNH₂

Threonine amide hydrochloride (400 mg) was suspended in 60%dimethylformamide (DMF) (2 ml). pH was adjusted to 6.5 with pyridine (20μl) and 6M NaOH (50 μl), Trypsin (100 mg) and DAI (100 mg). After 1/2hour the reaction was quenched by addition of formic acid (1 ml). Thereaction mixture was fractionated on "Sephadex G-50" with 1M aceticacid. The following fractions were collected:

    ______________________________________                                        Trypsin fraction:     60     mg                                               Insulin fraction:     102    mg                                               Remainder:            371    mg                                               ______________________________________                                    

The insulin fraction (102 mg) was purified by ion exchangechromatography on "DEAE-Sephadex A-25" equilibrated with 7M urea andeluted with a NaCl gradient according to EP 17938.

Pure human insulin amide (DAI--Thr--NH₂) in an amount of 59.2 mg(detected by amino acid analysis) was obtained.

Preparation of DAI--Thr (human insulin)

DAI--Thr--NH₂ (11 mg) was dissolved in 1 mM EDTA and 2 mM KCl (2 ml). pHwas adjusted to 9.0 by means of 0.5M NaOH (51 μl) and CPD-Y (50 μl, 13.6mg/ml) was added. After 15 min. the reaction was quenched with 6M HCl(25 μl) and the pH dropped to 1.2. The reaction mixture was separated on"Sephadex G-50" and eluted with 1M acetic acid. 8 mg of human insulinwas obtained. The amino acid analysis is shown in Table III below:

                  TABLE III                                                       ______________________________________                                        Amino acid analyses                                                           Human                                                                         insulin                            Human                                      (formula)    DAI           Amide   insulin                                    ______________________________________                                        Asp     3        2.97          3.05  3.04                                     Thr     3        1.91          2.94  2.90                                     Ser     3        2.86          2.86  2.95                                     Glu     7        7.27          7.23  7.04                                     Pro     1        0.94          0.95  0.85                                     Gly     4         4.000         4.000                                                                               4.000                                   Ala     1        1.00          1.00  1.04                                     Cys     6        5.31          5.19  5.02                                     Val     4        3.37          3.11  3.24                                     Ile     2        1.36          1.22  1.40                                     Leu     6        6.28          6.20  6.13                                     Tyr     4        3.64          3.97  3.77                                     Phe     3        2.82          2.92  2.83                                     His     2        1.96          1.96  1.87                                     Lys     1        0.94          1.00  0.95                                     NH.sub.3                                                                              6        8.12          6.46  6.98                                     Arg     1        0.98          0.99  0.87                                     Thr/Ser   1.00   1.003 (× 3/2)                                                                          1.027                                                                               0.984                                   ______________________________________                                    

REFERENCES

1. Ruttenberg, M.A.: Human insulin: Facile synthesis by modification ofporcine insulin, Science 177, 623-25(1972)

2. Gattner, H.-G., Schmitt, E. W., Naithani, V. K., in Semisyntheticpeptides and proteins, Eds. RE Offord and C-Di Bello, Academic Press,New York 1978, 181.

3. Obermeier, R., Geiger, R. (1976), Hoppe-Seyler's Z. Physiol. Chem.,357, 759-67.

4. Inouye, K., Watanabe, K., Morihara, K., Tochino, Y., Kanaya, T.,Emura, J.: Enzyme-assisted semisynthesis of human insulin, Journal ofthe American Chemical Society, (1979), vol. 101, 751-52.

5. Morihara, V.: Semi-synthesis of human insulin by trypsin-catalyzedreplacement of Ala-B30 by Thr in porcine insulin, Nature, Vol. 280, No.5721, 1979, 412-13.

6. Morihara, K., Oka, T., Tsuzuki, H., Tochino, Y., Kanaya, T.:Achromobacter protease I-catalyzed conversion of porcine insulin intohuman insulin, Biochemical and Biophysical Research Comm. Vol. 92, No.2, 1980, 396-402.

7. Widmer, F., Johansen, J. T.: Enzymatic peptide synthesiscarboxypeptides Y catalyzed formation of peptide bonds, Carlsberg Res.Commun, Vol. 44, Apr. 23, 1979, 37-46.

8. Johansen, J. T., Breddam, K., Ottesen, M.: Isolation ofCarboxypeptidase Y by affinity chromatography, Carlsberg Res. Commun.,Vol. 41, No. 1, 1976, 1-13.

9. Lee, H.-M., Riordan, J. F.: Does carboxypeptidase Y have intrinsicendopeptidase activity? Biochemical and Biophysical Research Comm., Vol.85, No. 3, 1978, 1135-1142.

10. Obermeier, Rainer: Des-octapeptide-(B23-30)-insulin, startingmaterial for human insulin and analogues; studies on synthesispurification and properties, Eds. RE Offord and C-Di Bello, AcademicPress, New York, 1978, 201-11.

11. Breddam, K., Widmer, F., Johansen, J. T.: Carboxypeptidase Ycatalyzed transpeptidations and enzymatic peptide synthesis, CarlsbergRes. Comm. Vol. 45, Nov. 4, 1980, p. 237-247.

12. Kubota et al. Carboxypeptidase C_(N), J. Biochem., Vol. 74, No. 4(1973), p. 757-770.

We claim:
 1. A process for enzymatic replacement of the B-30 amino acidin insulins, characterized by reacting as substrate component theselected insulin Ins-X, wherein X represents the B-30 amino acid,with anamine component selected from the group consisting of (a) optionallyN-substituted amino acid amides of the formula

    H--B--NR.sup.1 R.sup.2

wherein B is an amino acid residue and R¹ and R² are independentlyselected from the group consisting of hydrogen, amino, hydroxy, alkyl,cycloalkyl, aryl, heteroaryl and aralkyl or R¹ and R² together with thenitrogen atom form a heterocyclic group which may contain a furtherheterd atom, and (b) amino acid esters of the formula

    H--B--OR.sup.3, H--B--SR.sup.3 or H--B SeR.sup.3

wherein B is an amino acid residue and R³ represents alky, cycloalkyl,aryl, heteroaryl or aralkyl,in the presence of an L-specific serine orthiol carboxypeptidase enzyme in an aqueous solution or dispersionhaving a pH from about 7 to 10.5, thereby to form an insulin derivativeIns--B--NR¹ R², Ins--B--B--NR¹ R², Ins--B--OR³, Ins--B--SR³ orIns--B--Ser³.
 2. The process according to claim 1, characterized byusing as the carboxypeptidase enzyme a serine or thiol carboxypeptidasefrom yeast or of animal, vegetable or microbial origin.
 3. The processaccording to claim 2, characterized by using carboxypeptidase Y fromyeast as the carboxypeptidase enzyme.
 4. The process according to claim3, characterized by using a carboxypeptidase Y which has been purifiedby affinity chromatography on an affinity resin comprising a polymericresin matrix with a plurality of coupled benzylsuccinyl groups.
 5. Theprocess according to claim 2, characterized by using a carboxypeptidaseenzyme selected from the group consisting of penicillocarboxypeptidaseS-1 and S-2 from Penicillium janthinellum, carboxypeptidases fromAspergillus saitoi or Aspergillus oryzae, carboxypeptidases C fromorange leaves or orange peels, carboxypeptidase C_(N) from Citrusnatsudaidai Hayata, phaseolain from french bean leaves,carboxypeptidases from germinating barley, germinating cotton plants,tomatoes, watermelons and Bromelain(pineapple)powder.
 6. The processaccording to claim 1, characterized by using an immobilizedcarboxypeptidase enzyme.
 7. The process according to claim 1,characterized by maintaining the pH in the solution or dispersion at 7.5to 10.5.
 8. The process according to claim 1, characterized by using anorganic solvent selected from the group consisting of alkanols, dimethylsulfoxide, dimethyl formamide, dioxane, tetrahydrofurane, dimethoxyethane, ethylene glycol and polyethylene glycols.
 9. The processaccording to claim 1, characterized by using an aqueous solution ordispersion containing urea or guanidine hydrochloride in a concentrationof up to 3 molar.
 10. The process according to any of claims 1, or 2,characterized in that the substrate component is porcine insulin and theamino acid forming part of the amine component is threonine, the aminecomponent being threonine amide.
 11. The process of claim 1 furthercomprising cleaving from said insulin derivative a group --NR¹ R²,--B--NR¹ R², --OR³, --SR³, or SeR³.
 12. The process according to claim11 characterized in that the group --NR¹ R², --B--NR¹ R², --OR³, --SR³or --SeR³ is removed by treating said insulin derivative with anL-specific serine or thiol carboxypeptidase enzyme from yeast or ofanimal, vegetable or microbial origin in an aqueous solution ordispersion having a pH from about 7 to about 10.5.